Mitochondrial dysfunction is associated with several human diseases, including neurodegenerative diseases, type 2 diabetes, cardiovascular disease, myopathies and deafness. It is also thought to underlie the process of ageing and ageing-associated pathologies. The orphan nuclear receptors ERR1, ERR2 and ERR3 (Estrogen-Related Receptors), in co-operation with transcriptional coactivators of the PGC-1 family, regulate the expression of genes promoting mitochondrial biogenesis, oxidative metabolism and protection from oxidative stress. Accordingly, mechanisms that enhance the activity of ERR and/or PGC-1 proteins have potential applications in the design of strategies for enhancing mitochondrial function and energy metabolism pathways and ameliorating pathologies associated with mitochondrial dysfunction. Agonist ligands of nuclear receptors typically enhance receptor activity by inducing conformational changes that promote cooperation with transcriptional coactivators. Thus, agonist of ERRs would be predicted to increase cooperation with PGC-1 coactivators and thereby enhance the expression of genes important for mitochondrial function and oxidative metabolism. Currently, we know of no small molecule probes that act as ERR agonists in the presence of PGC-1 coactivators. The proposed work will take advantage of recent molecular advances in our understanding of ERR regulation to: 1) develop a robust high-throughput cell- based assay tailored specifically to ERR biology and suitable for identifying ERR3 agonists that enhance ERR3 activity in the presence of PGC-11;2) establish a series of counter- and secondary assays that will be used to determine specificity and efficacy of identified probes;3) adapt the assay to high throughput screening (HTS) format suitable for screening of a compound collection at a NIH MLSCN screening center;and 4) perform a pilot screen to identify agonists of ERR3. In the long-term, through a larger screening effort, we expect to identify probes that enhance the functional interaction of ERR3 with the transcriptional coactivator PGC-11 and/or other ERR3 coactivators, (i.e. ERR3 agonists), and develop probe derivatives with improved potency, efficacy, selectivity and bioavailability. These improved probes will eventually be used to a) pharmacologically dissect the physiological functions of ERR2 and ERR3 in vivo in mouse models, and b) modulate mitochondrial function and energy metabolism pathways and possibly ameliorate pathologies associated with mitochondrial dysfunction. PUBLIC HEALTH RELEVANCE: Mitochondrial dysfunction is associated with several human diseases, (including neurodegenerative diseases, type 2 diabetes, cardiovascular disease, myopathies and deafness), and is thought to underlie the process of ageing. The orphan nuclear receptors " Estrogen-Related Receptors" (ERRs) control the expression of genes important for the generation and proper function of mitochondria. Thus, identification of small molecule probes that activate ERRs is an important step towards the design of dugs that may enhance mitochondrial function and have applications in diseases and syndromes of mitochondrial dysfunction.